Rhesus BCRP and antibodies thereto

ABSTRACT

The present invention provides a breast cancer resistance polynucleotide and protein of  Macaca mulata  (rhesus monkey) and uses thereof. The present invention also provides anti-BCRP antibodies and uses thereof, particularly as a hematopoietic stem cell marker.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/644,706, filed Jan. 18, 2005, which is hereby incorporated byreference its entirety.

BACKGROUND OF THE INVENTION

Rhesus monkeys (Macaca mulata) have been utilized extensively as apre-clinical model in hematopoietic stem cell transplant research. Inorder to carry out such research, it is important to be able to identifyand isolate the hematopoietic stem cells from rhesus bone marrow orperipheral blood. Recently, BCRP has been focused upon as a marker forthe very primitive hematopoietic or other organ stem cells.

Stem cells have the capacity to become at least all differentiated celltypes of their lineage in that tissue. Stem cells have two importantcharacteristics that distinguish them from other types of cells. First,they are unspecialized cells that renew themselves for long periodsthrough cell division. Secondly, under suitable conditions they can beinduced to become cells with special functions, which may be considereddifferentiated.

Stem cells have been identified in a variety of tissues. They can bedistinguished by a variety of means, such as by the tissue from whichthey were harvested, their bias in differentiation ability, the stage ofdevelopment at which they exist, and their gene expression profile. Inparticular, stem cells may be from ectoderm (epidermal, neural, neuralcrest, and hair follicle); mesoderm (cardiac muscle, skeletal muscle,umbilical cord blood, mesenchymal, hematopoietic, umbilical cord matrix,and multipotent adult precursor); endoderm (pancreatic islet and hepaticoval); and germ (primordial germ) stem cells. More than one stem cellmay be present in a particular tissue. For example, in the hematopoieticsystem alone, there are stem cells from the yolk sac, fetal cord blood,liver, and adult bone marrow.

Although stem cells may be derived from any tissue harboring stem cells,in particular embodiments they are from bone marrow, embryos,mesenchyme, neural tissue, pancreatic tissue, muscle tissue (such ascardiac muscle), liver, skin, intestine, nasal epithelium, bone,pancreas, or germ cells, for example.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 (A-C) shows an amino acid comparison between monkey and humanBCRP. BRCP (SEQ ID NO:2); gi|74136389|ref (NP_(—)00102809; Macacamulatta; 654 amino acids; SEQ ID NO:5); gi|62526033|ref (NP_(—)004818;Homo sapiens; 655 amino acids; SEQ ID NO:6); gi|4185796|gb|A (AAD09188;Homo sapiens; 655 amino acids; SEQ ID NO:7); gi|27450414|gb| (AAO14617;Homo sapiens; 655 amino acids; SEQ ID NO:8).

DESCRIPTION OF THE INVENTION

The present invention relates to all facets of rhesus monkey nucleicacids encoding breast cancer resistance protein, including polypeptidesencoded by the nucleic acids, antibodies and specific binding partnersthereto, and their applications to research, diagnosis, drug discovery,therapy, clinical medicine, forensic science and medicine, etc. Thepolynucleotides and polypeptides are useful in variety of ways,including, but not limited to, as molecular markers (e.g., for stemcells), as selectable markers (e.g. based on their drug resistanceactivity), as drug targets, and for detecting, diagnosing, staging,monitoring, prognosticating, preventing or treating, determiningresistance to, etc., diseases and conditions.

Breast cancer resistance protein (“BCRP”) is an ATP-binding cassette(ABC) transporter gene that is expressed in various tissues, includingplacenta. It is also known as ATP-binding cassette, subfamily G, member2 (“ABCG2”). The protein is related to the Drosophila white and yeastADP 1 genes, and is a member of a subfamily that includes severalmultidrug resistance transporters. Human BCRP was cloned from amultidrug-resistant human breast cancer cell culture line that displayedan ATP-dependent reduction in the intracellular accumulation ofanthracycline anticancer drugs. Expression of the full-length BCRP cDNAin MCF-7 human breast cancer cells conferred resistance to mitoxantrone,doxorubicin, and daunorubicin, reduced daunorubicin accumulation andretention, and caused an ATP-dependent enhancement of the efflux orrhodamine-123 in the cloned transfected cells. See, e.g., Doyle et al.,Proc. Nat. Acad. Sci. 95: 15665-15670, 1998. Thus, polypeptides andpolynucleotides of the present invention can be utilized diagnosticallyto determine the molecular basis for drug diseases and conditionsassociated with drug resistance, especially cancer in which theresistance has been acquired.

As explained in more detail below, the present invention also providespolynucleotides, polypeptides, and antibodies thereto, which arespecific to rhesus monkey BCRP, especially antibodies which are specificand do not cross-react with a human BCRP.

Polynucleotides

A polynucleotide encoding a rhesus monkey (Macaca mulatta) BCRP has beenisolated from pancreatic islet cells. The present invention relates tothis specific polynucleotide sequence (SEQ ID NO:1;), especially to itscoding sequence (from 34-2001 of SEQ ID NO:1), polynucleotides whichcode for the BRCP polypeptide (SEQ ID NO:2), and derivatives andfragments thereof, including polynucleotides which hybridize to thecomplement of SEQ ID NO: 1 under high stringency conditions. Thisincludes polynucleotides which code for BCRP and which possess or moreactivities of BCRP, including, ATPase activity, drug transport activity(e.g., mitoxantrone, topotecan, flavopiridol, and daunorubicin), andATP-binding activity. Assays for these activities can be carried outroutinely. See, e.g., Ozvegy et al., J. Biol. Chem., 277:47980-47990,2002. For example, the present invention provides a polynucleotidesequence comprising a nucleotide sequence which hybridizes to thecomplement of SEQ ID NO:1 under high stringency conditions and whichcodes for a polypeptide having an ATPase and drug transport activity.The coding sequence of such polynucleotide can comprise about 90%, 92%,96%, 97%, 98%, 99%, or more nucleotide sequence identity along itsentire length to the entire length of SEQ ID NO:1. The present inventionalso provides fragments of such polynucleotides, including fragmentshaving one or more of the above-mentioned activities, including as wellthe ability to elicit antibodies specific to BCRP. Fragments cancomprise, e.g., 20 nucleotide, 40 nucleotides, 50 nucleotides, 57nucleotides, 60 nucleotides, or more nucleotides, etc.

The present invention also provides polynucleotides comprising,consisting of, consisting essentially of (e.g., having 25 or less, 20 orless, 15 or less, 10 or less, 5 or less, additional polynucleotides atits 5′ and/or 3′ terminus) a polynucleotide of SEQ ID NO:3, or apolynucleotide coding for SEQ ID NO:4. The present invention alsoprovides polynucleotides which have at least 90%, 95%, 96%, 97%, 98%,99% or more identity to SEQ ID NO:3, to a polynucleotide coding for SEQID NO:4, or to polynucleotides which hybridize to the complement of SEQID NO:3 under high stringency conditions. Such polynucleotides can beprepared and/or isolated routinely.

A polynucleotide according to the present invention can be obtained froma variety of different sources. It can be obtained from DNA or RNA, suchas polyadenylated mRNA or total RNA, e.g., isolated from tissues, cells,or whole organism. The polynucleotide can be obtained directly from DNAor RNA, from a cDNA library, from a genomic library, etc. Thepolynucleotide can be obtained from a cell or tissue (e.g., from anembryonic or adult tissues) at a particular stage of development, havinga desired genotype, phenotype, disease status, etc. A polynucleotidewhich “codes without interruption” refers to a polynucleotide having acontinuous open reading frame (“ORF”) as compared to an ORF which isinterrupted by introns or other noncoding sequences, e.g., in the formwhen isolated from a cDNA library.

A polynucleotide of the present invention can comprise additionalpolynucleotide sequences, e.g., sequences to enhance expression,detection, uptake, cataloging, tagging, etc. A polynucleotide caninclude only coding sequence; a coding sequence and additionalnon-naturally occurring or heterologous coding sequence (e.g., sequencescoding for leader, signal, secretory, targeting, tagging, enzymatic,fluorescent, antibiotic resistance, and other functional or diagnosticpeptides); coding sequences (e.g., an initiation codon or a leadersequence) and non-coding sequences, e.g., untranslated sequences ateither a 5′ or 3′ end, or dispersed in the coding sequence, e.g.,introns.

A polynucleotide according to the present invention also can comprise anexpression control sequence operably linked to a polynucleotide asdescribed above. The phrase “expression control sequence” means apolynucleotide sequence that regulates expression of a polypeptide codedfor by a polynucleotide to which it is functionally (“operably”) linked.Expression can be regulated at the level of the mRNA or polypeptide.Thus, the expression control sequence includes mRNA-related elements andprotein-related elements. Such elements include promoters, enhancers(viral or cellular), ribosome binding sequences, transcriptionalterminators, etc. An expression control sequence is operably linked to anucleotide coding sequence when the expression control sequence ispositioned in such a manner to effect or achieve expression of thecoding sequence. For example, when a promoter is operably linked 5′ to acoding sequence, expression of the coding sequence is driven by thepromoter. Expression control sequences can include an initiation codonand additional nucleotides to place a partial nucleotide sequence of thepresent invention in-frame in order to produce a polypeptide (e.g., pETvectors from Promega have been designed to permit a molecule to beinserted into all three reading frames to identify the one that resultsin polypeptide expression). Expression control sequences can beheterologous or endogenous to the normal gene.

A polynucleotide of the present invention can also comprise nucleic acidvector sequences, e.g., for cloning, expression, amplification,selection, etc. Any effective vector can be used. A vector is, e.g., apolynucleotide molecule which can replicate autonomously in a host cell,e.g., containing an origin of replication. Vectors can be useful toperform manipulations, to propagate, and/or obtain large quantities ofthe recombinant molecule in a desired host. A skilled worker can selecta vector depending on the purpose desired, e.g., to propagate therecombinant molecule in bacteria, yeast, insect, or mammalian cells. Thefollowing vectors are provided by way of example. Bacterial: pQE70,pQE60, pQE-9 (Qiagen), pBS, pD10, Phagescript, phiX174, pBK Phagemid,pNH8A, pNH16a, pNH18Z, pNH46A (Stratagene); Bluescript KS+II(Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).Eukaryotic: PWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, PBPV,PMSG, pSVL (Pharmacia), pCR2.1/TOPO, pCRII/TOPO, pCR4/TOPO, pTrcHisB,pCMV6-XL4, etc. However, any other vector, e.g., plasmids, viruses, orparts thereof, may be used as long as they are replicable and viable inthe desired host. The vector can also comprise sequences which enable itto replicate in the host whose genome is to be modified.

Polynucleotides which hybridize to polynucleotides of the presentinvention can be selected in various ways. Filter-type blots (i.e.,matrices containing polynucleotide, such as nitrocellulose), glasschips, and other matrices and substrates comprising polynucleotides(short or long) of interest, can be incubated in a prehybridizationsolution (e.g., 6×SSC, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA,5× Denhardt's solution, and 50% formamide), at 22-68° C., overnight, andthen hybridized with a detectable polynucleotide probe under conditionsappropriate to achieve the desired stringency. In general, when highhomology or sequence identity is desired, a high temperature can be used(e.g., 65° C.). As the homology drops, lower washing temperatures areused. For salt concentrations, the lower the salt concentration, thehigher the stringency. The length of the probe is another consideration.Very short probes (e.g., less than 100 base pairs) are washed at lowertemperatures, even if the homology is high. With short probes, formamidecan be omitted. See, e.g., Current Protocols in Molecular Biology,Chapter 6, Screening of Recombinant Libraries; Sambrook et al.,Molecular Cloning, 1989, Chapter 9.

For instance, high stringency conditions can be achieved by incubatingthe blot overnight (e.g., at least 12 hours) with a polynucleotide probein a hybridization solution containing, e.g., about 5×SSC, 0.5% SDS, 100μg/ml denatured salmon sperm DNA and 50% formamide, at 42° C., orhybridizing at 42° C. in 5×SSPE, 0.5% SDS, and 50% formamide, 100 μg/mldenatured salmon sperm DNA, and washing at 65° C. in 0.1% SSC and 0.1%SDS. Blots can be washed at high stringency conditions that allow, e.g.,for less than 5% bp mismatch (e.g., wash twice in 0.1% SSC and 0.1% SDSfor 30 min at 65° C.), e.g., selecting sequences having 95% or greatersequence identity.

Polypeptides

The present invention provides polypeptides having an amino acidsequence of SEQ ID NO:2, fragments thereof, and derivatives thereof. Anexample of a fragment of SEQ ID NO:2 is a polypeptide which consists ofNYATCTGEEYLTKQGIDLS (SEQ ID NO:4) which is amino acids 604-622 of SEQ IDNO:2 (e.g., an extracellular region). Also included isAcetyl-NYATCTGEEYLTKQGIDLS-amide (SEQ ID NO:4). Such fragment can beutilized to generate antibodies which are specific to rhesus monkeyBCRP, especially a polypeptide of SEQ ID NO:2, and which do notcross-react (i.e., specifically bind to) human BCRP. Such lack ofcross-reactivity can be measured when the BCRP polypeptide is expressedin a cell membrane in its normal configuration.

The present invention also provides polypeptides comprising, consistingof, and consisting essentially of (e.g., having 25 or less, 20 or less,15 or less, 10 or less, 5 or less, etc., additional amino acids at itsN- and C-terminus) a polypeptide of SEQ ID NO:4. The present inventionalso provides polypeptides which have at least 90%, 92% 95%, 96%, 97%,98%, 99%, etc., or more identity to SEQ ID NO:4. Such polypeptides canbe prepared and/or isolated routinely.

The present invention also provides an isolated polypeptide comprising:a) a polypeptide having an amino acid sequence of SEQ ID NO:2, or apolypeptide fragment thereof; or b) a polypeptide which is encoded by apolynucleotide sequence which hybridizes to the complement of SEQ IDNO:1 under high stringency conditions, or a polypeptide fragmentthereof, wherein said polypeptide of (a) and (b) is capable of elicitingantibodies which specifically bind to a rhesus monkey BCRP of SEQ IDNO:2.

High stringency conditions can be determined routinely, e.g., to selectpolynucleotides having at least about 90%, 95%, etc., identity to thecomplement of SEQ ID NO:2 or SEQ ID NO:4. Specific hybridizationconditions are described above. The polypeptides encoded bypolynucleotides isolated in such manner are “capable of elicitingantibodies which specifically bind to a rhesus monkey BCRP.” By thelater phrase, it is meant that, when the polypeptide is used as animmunogen, e.g., in a mouse or rabbit, it produces an immune responsethat includes antibodies specific to BCRP. Specific binding to BCRP canbe determined routinely, e.g., as described below. In addition,antibodies, especially polyclonal antibodies, can be fractionated toseparate fractions have a desired specificity, e.g., to a particularregion of the polypeptide.

The present invention provides polypeptides which comprise at least onerhesus monkey isoform-specific residue, e.g., selected from the groupconsisting of: T242, P340, K357, G411, and T615 of SEQ ID NO: 2. Thelatter nomenclature (e.g., “T242”) as used herein indicates the aminoacid and residue position at which the amino acid is present (e.g., T242indicates a threonine is present at amino acid position 242 of SEQ IDNO:2).

The term “monkey isoform-specific residue” refers to the amino acidresidues which are specific to the monkey isoform of SEQ ID NO:2, and,e.g., not present in human BCRP of SEQ ID NOS: 6, 7, and 8; and, e.g.,not present in rhesus monkey BCRP of SEQ ID NO: 5 which represents arhesus monkey isoform different from SEQ ID NO:2. FIG. 1 shows an aminoacid alignment of rhesus monkey and human BCRP from which these andother position numbers can be determined.

The present invention also provides polypeptides which comprise at leastone rhesus monkey-specific residue selected from the group consistingof: M12, T22, R28, V48, G55, P149, V329, D338, I404, G411, N417, F471,T502, I508, V599, and T602. A monkey-specific residue is an amino acidwhich is specific to a rhesus monkey BCRP (e.g., SEQ ID NO:2), but notpresent in the human homolog. FIG. 1 shows an amino acid alignment ofrhesus monkey and human BCRP from which these and other position numberscan be determined.

The present invention also provides polypeptides which have amino acidsubstitutions other than those at the indicated position number in FIG.1, especially at amino acid positions 242, 340, 357, 411, and/or 615 ofSEQ ID NO:2; or 12, 22, 28, 48, 55, 149, 329, 338, 404, 411, 417, 471,502, 508, 599, and/or 602 of SEQ ID NO:2. Conservative andnon-conservative amino acid substitutions can be made. Conservativeamino acid substitutions include exchanges within one of the followingfive groups: I. Small aliphatic, nonpolar or slightly polar residues:Ala, Ser, Thr, Pro, Gly; II. Polar, negatively charged residues andtheir amides: Asp, Asn, Glu, Gln; III. Polar, positively chargedresidues: His, Arg, Lys; IV. Large, aliphatic, nonpolar residues: MetLeu, Ile, Val, Cys; V. Large, aromatic residues: Phe, Tyr, Trp.

Preferred polypeptides include fragments which contain T615 of SEQ IDNO:2, such as SEQ ID NO:4. Preferred antibodies, or fragment thereof,include antibodies which specifically bind to an epitope present inthese polypeptides, especially where the epitope comprises T615.

The polypeptides of the present invention, especially fragments of thepolypeptide having an amino acid sequence of SEQ ID NO:2, such as afragment having the amino acid sequence set forth in SEQ ID NO:4, canalso be conjugated or fused to other proteins, such as carrier proteins(e.g., for vaccination purposes) or at its N- or C-terminus forrecombinant production, such as for stabilizing the polypeptide or forpurification purposes after recombinant expression. Examples ofpolypeptides that can be fused recombinantly to a polypeptide (e.g., toa polypeptide of SEQ ID NO:4) of the present invention includes, e.g.,His, Myc, HA, GST, VSV, beta-gal, lac, GFP, thioredoxin, MBP, and other“tag” sequences which can be used to purify the polypeptide afterexpression in a host cell. Commercial kits can be used to clone, express(in both eukaryotic and prokaryotic hosts), and purify polypeptideswhich comprise these polypeptide tags. Expression can be achieved in E.coli, yeast, baculovirus, and mammalian systems to produce large amountsof recombinant proteins that may otherwise be difficult to isolate fromnatural cells and tissues. Antibodies to the fusion proteins (e.g., SEQID NO:4 fused to a tag) can be used to monitor protein expression andpurification. The peptide tags have their own characteristics which canbe employed for purification purposes. Poly-His-fusion proteins (6×His)can bind to Nickel-Sepharose or Nickel-HRP. GST-fusion proteins can bindto glutathione-Sepharose. Therefore, a high degree of purification offusion protein can be achieved in just one affinity purification step.Fusion proteins can be directly injected into animals to generateantibodies, or the peptide tags can be removed by treatment with enzymesand other cleavage agents to generate tag-free recombinant proteins. Thefusion proteins can be produced recombinantly, e.g., by fusing in-framea polynucleotide coding for the tag sequence to a polynucleotide codingfor a BCRP polypeptide (either to its N- or C-terminus).

Amino acid comparisons are shown in FIG. 1 between the BCRP of thepresent invention and other related BCRP polypeptides. Sequence identitybetween SEQ ID NO:2 and NP_(—)001028091 (Macaa mulatta) is 650/655(99%); between NP-004818 (human) is 631/655 (96%); between AAD09188(human) is 629/655 (96%); and between AAO14617 is 630/655 (96%). Asindicated above, the isoform of SEQ ID NO:2 contains certain uniqueamino acid substitutions in comparison to other known forms, and thepresent invention provides polypeptides which comprise one or more ofthe following amino acid residues, e.g., T242, P340, G411, and/or T615.

Binding Partners

The polynucleotides and amino acid sequences provided herein are usefulfor generating antibodies, and other binding partners, against the cellsurface membrane polypeptide BCRP (SEQ ID NO:2) in rhesus and othermonkeys. Rhesus has been used as a model for hematopoietic stem celltransplantation, and expression of BCRP is known to serve as a markerfor primitive hematopoietic stem cells. Therefore, the antibodycompositions of the present invention can be used for the detection ofBCRP expression in hematopoietic stem cells. A polypeptide comprisingSEQ ID NO:4 can be utilized to produce antibodies which are specific toa rhesus BCRP of the present invention. The antibodies can be selectedsuch that they specifically recognize (bind) rhesus BCRP, but not ahuman BCRP, especially when the polypeptide is displayed on the cellsurface or on a cell membrane. Such an antibody can recognize an epitopethat comprises, e.g., at least one of T242, P340, K357, G411, and/orT615, especially T615, of SEQ ID NO:2. An antibody can also recognize anepitope comprising at least one of M12, T22, R28, V48, G55, P149, V329,D338, I404, G411, N417, F471, T502, I508, V599, and T602 of SEQ IS NO:2.

Binding partners include, e.g., antibodies, aptamers, and other bindingligands. Antibodies can be of any type, including, e.g., polyclonal,monoclonal, recombinant, chimeric, humanized, single-chain, Fab, andfragments thereof. These can be prepared routinely according to anydesired method. See, also, screening recombinant immunoglobulinlibraries (e.g., Orlandi et al., Proc. Natl. Acad. Sci., 86:3833-3837,1989; Huse et al., Science, 256:1275-1281, 1989); in vitro stimulationof lymphocyte populations; Winter and Milstein, Nature, 349: 293-299,1991. See, also, U.S. Pat. No. 5,260,203 for methods of making andproducing single-chain antibodies (e.g., “ScFv”) The antibodies can beIgM, IgG, subtypes, IgG2a, IgG1, etc. An antibody specific for apolypeptide (e.g., a polypeptide having the amino acid sequence setforth in SEQ ID NO:4) means that the antibody recognizes and binds to adefined sequence of amino acids within or including the polypeptide.

The term “antibody” as used herein includes intact molecules as well asfragments thereof, such as Fab, F(ab′)2, Fv, and single chainantibodies, which are capable of binding to an epitopic determinantpresent in a BCRP polypeptide. Such antibody fragments retain theability to specifically bind to a BCRP epitope. The term “epitope”refers to an antigenic determinant on an antigen to which the paratopeof an antibody binds. Epitopic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three-dimensional structuralcharacteristics, as well as specific charge characteristics. Antibodiescan be prepared against specific epitopes or polypeptide domains.

Various modifications can be made to the polypeptides and antibodies ofthe present invention, such as attaching detectable markers (e.g.,avidin, biotin, enzymes, radioactive elements, fluorescent tags anddyes, energy transfer labels, energy-emitting labels, binding partners,etc.) or moieties which improve detection, and/or stability (e.g., PEG).Examples of fluorescent dyes include, but are not limited to, AlexaFluor 35, 488, 532, 546, 555, 568, 594, 647, 660, and 680; Fluorescein(FITC); SpectrumGreen; Rhodamine 6G; tetramethylrhodamine (TRITC);SpectrumOrange; Lissamine rhodamine B dye; Texas Red dye, SpectrumRed,PE; APC; Cy5; Cy5.5; Cy7; quantum dots; fluorescent microspheres (seee.g., U.S. Pat. No. 5,786,219), including spheres with magneticproperties (e.g., U.S. Pat. Application No. 20010046602); etc. Examplesof enzymes include, e.g., peroxidases and others as mentioned below.

Polyclonal antibodies to the polypeptides can generally be raised inanimals by multiple subcutaneous (sc) or intraperitoneal (ip) injectionsof the polypeptide and an adjuvant. It may be useful to conjugate thepolypeptide to a carrier, especially a carrier which is immunogenic inthe species to be immunized, e.g., keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin, soybean trypsin inhibitor, or tetanustoxoid. Conjugation can be carried routinely, e.g., using a bifunctionalor derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, etc.

The route and schedule for immunization of the host animal are generallyin keeping with established and conventional techniques for antibodystimulation and production. Any mammalian subject can be used togenerate antibodies, including rabbits, mice, rats, guinea pigs, sheep,goats, and antibody-producing cells obtained from them. Animals can beimmunized routinely, e.g., using immunogenic conjugates, or derivativesthereof, by combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freund's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later, theanimals can be boosted with about 1/20 (or less) to 1/10 the originalamount of conjugate in Freund's complete adjuvant (or other suitableadjuvant) by subcutaneous injection at multiple sites. 7 to 14 dayslater animals can be bled and the serum can be assayed for antibodytiter. Animals can be boosted until the titer plateaus.

After immunization, monoclonal antibodies can be prepared by recoveringimmune lymphoid cells, such as spleen cells or lymphocytes from lymphnode tissue, from immunized animals and immortalizing the cells in aconventional fashion, e.g., by fusion with myeloma cells or byEpstein-Barr (EB)-virus transformation and screening for clonesexpressing the desired antibody. The hybridoma technique describedoriginally by Kohler and Milstein, Eur. J. Immunol. 6:511 (1976) hasbeen widely applied to produce hybrid cell lines that secrete highlevels of monoclonal antibodies against many specific antigens. It ispossible to fuse cells of one species with another. However, typicallythe source of the immunized antibody producing cells and the myeloma arefrom the same species.

The hybrid cell lines can be maintained in culture in vitro in cellculture media. The cell lines of this invention can be selected and/ormaintained in a composition comprising the continuous cell line inhypoxanthine-aminopterin thymidine (HAT) medium. Hybridoma cell linescan be stored and preserved in any number of conventional ways,including freezing and storage under liquid nitrogen. Frozen cell linescan be revived and cultured indefinitely with resumed synthesis andsecretion of monoclonal antibody. The secreted antibody can be recoveredfrom tissue culture supernatant by conventional methods, includingprecipitation, ion exchange chromatography, affinity chromatography,etc. The antibodies described herein can also be recovered fromhybridoma cell cultures by conventional methods for purification of IgGor IgM as the case may bethat heretofore have been used to purify theseimmunoglobulins from pooled plasma, e.g., ethanol or polyethylene glycolprecipitation procedures.

Antibodies, and immune responses, can also be generated by administeringnaked DNA See, e.g., U.S. Pat. Nos. 5,703,055; 5,589,466; and 5,580,859.

Antibodies in accordance with the present invention can also be preparedby recombinant techniques. These include methods for creatingrecombinant DNA versions of the antigen-binding regions of antibodymolecules (known as Fab or variable regions fragments) which bypass thegeneration of monoclonal antibodies. For example, antibody genes fromsource cells (including naïve cells and cells expressing the antibody ofinterest) can be cloned into an appropriate vector and utilized toachieve expression of adequate amounts of functional antibody.Additional techniques can be utilized to select the desired antibodywhen a large repertoire of genes has been cloned. Recombinant antibodiescan be cloned from any species of antibody-producing animal using theappropriate oligonucleotide primers.

The ability to clone antibody genes makes it possible to generate newantibodies in vitro which have increased binding specificity for thetarget polypeptide, e.g., using affinity maturation techniques. This canbe done in various ways, including at the level of the whole combiningsite by making new combinations of H and L chains (Collet et al., 1992;Kang et al., 1991a; Marks et al., 1992). It can also be done by mutatingindividual CDRs (Cheetham, 1988; Garrard and Henner, 1993; Kettleboroughet al., 1991).

The host in which most recombinant antibody methods were originallydeveloped is the bacterium Escherichia coli and other bacterial hosts.Growth of bacteria is rapid and inexpensive, and a number of vectors areavailable for expression and manipulation of cloned genes. DNA can beintroduced directly into E. coli (the process known as transformation)or by infectious bacteriophage (transfection). Genetic constructions ofantibody fragments (Fab and ScFv) can be quickly assessed and variousselection methods can be applied.

Phage selection methods can also be used to prepare recombinantantibodies of the present invention. Phage displaying the desiredantibodies can be selected by binding to antigen in a format similar tosolid-phase immunoassay (Barbas and Lerner, 1991). The process isgenerally called “phage panning.” The antigen or hapten conjugate isimmobilized on microplate wells or on magnetic beads or solid materialpacked into a column. Bound phage can be eluted and amplified byreplication in new host cells. Those that bind weakly or not at all arewashed away before the elution step. After several rounds of binding andamplification, the phage population should consist almost entirely ofthose that express the desired antibodies.

Antibody engineering, the process of altering antibody structure andfunctional properties by recombinant DNA methods, can also be applied tothe antibodies of the present invention. Once the DNA sequences of thevariable regions are known, the amino acid sequence can be deduced.Methods of in vitro mutagenesis can be applied to insert, delete, orchange one or several amino acids, or to exchange entire variabledomains. The structures of many antibodies have been determined by X-raycrystallography at atomic resolution, and the coordinates are stored asfiles in the Brookhaven Protein Data Bank. These files may be retrievedthrough the Internet and displayed in many programs available onminicomputers and modeling workstations. Because there is considerablehomology among antibody framework domains and the secondary structuresof some CDRs, it is possible to construct a computational model of a newantibody in order to predict which CDRs and other residues are importantto epitope binding. The model can then be used to guide subsequentengineering and mutagenesis steps (Roberts et al., 1994). The softwarepackage AbM (Oxford Molecular, Ltd.) uses established crystallographicstructures to build antibody models from amino acid sequence data. Theresulting model, which consists of a set of atomic coordinates inthree-dimensional space, can be compared to known antibody structures.The mutations can then be introduced into the antibody sequences in thecloning vector, expressed, and assayed routinely for binding affinityand characteristics.

Affinity and specificity, or both, can be modified and improved bychanging the relative orientations of VH and VL domains at theirinterface, lengthening or shortening particular CDRs to enlarge orshrink the binding pocket, increasing the flexibility of CDRs in thecombining site, removing or re-spacing some of the side chains that formthe combining site, or altering residues that do not contact antigen buthelp to form the combining site through CDR-CDR and CDR-frameworkinteractions (Roberts et al., 1987). The structure can be changed toaccommodate water molecules, metal atoms, and other functional groups.In principle other properties of the antibody-antigen interactions canbe changed by altering close-contact residues. The antibody can also befused with other antibody molecules, toxins, or enzymes.

Synthetic combinatorial antibody libraries have been developed as analternative to the use of animals for antibody production (Barbas etal., 1992; Hoogenboom and Winter, 1992; Lerner et al., 1992). Based onthe knowledge that antigen binding is due primarily to interactions withthe six CDR loops in the combining site, antibody diversity can begenerated in vitro by limited mutagenesis of one or more CDRs. Such alibrary can be orders of magnitude more diverse than the 10⁵ to 10⁶antibodies expressed by the mammalian genome. A library can be displayedon the surface of phage and new antibodies selected by panning asdescribed previously.

Combinatorial library design is based on the unique structuralsimilarities in all antibodies, irrespective of their bindingspecificity. CDRH3 has the greatest flexibility and conformationalvariability, and may have the greatest influence on antigen binding. Oneof the first combinatorial Fab libraries was constructed by makingsequence and length variations in CDRH3 of a human Fab specific fortetanus toxoid (Barbas et al., 1992). It is also known that amino acidside chains on at least five of the six CDRs generally contact theepitope (Roberts et al., 1993; Wilson and Stanfield, 1993). Consequentlyother combinatorial libraries have been constructed by introducingdiversity in several of the CDRs (Garrard and Henner, 1993). Anotherstrategy made use of the natural diversity of ScFv libraries preparedwith CDRs from the immunologically naive germline antibody genes(Griffiths et al., 1993; Marks et al., 1991). A third approach togenerating diversity seeks to improve the affinity of selectedantibodies by allowing random or directed reassortment of cloned VH andVL regions (Marks et al., 1992). This type of diversity has been called“chain shuffling.” Another recently published method allowscombinatorial shuffling of individual intact CDRs (Crameri and Stemmer,1995).

Methods of Detecting Antibodies and Polypeptides

The present invention also provides methods of detecting a rhesus monkeyBCRP polypeptide, comprising one or more of the following steps in anyeffective order, e.g., contacting a sample comprising a rhesus monkeyBCRP polypeptide with an antibody specific for it under conditionseffective for said antibody to specifically bind to said polypeptide;and detecting said specific binding of said antibody to said BCRPpolypeptide. Specific binding of antibodies and other binding partnersto BRCP polypeptides, or fragments thereof, can be detected, visualized,determined, quantitated, etc. according to any effective method. Usefulmethods include, e.g., but are not limited to, immunoassays, RIA(radioimmunassay), ELISA, (enzyme-linked-immunosorbent assay),immunofluorescence, flow cytometry (e.g., fluorescence activated cellsorting or FACS), histology, electron microscopy, light microscopy, insitu assays, immunoprecipitation, Western blot, etc. Antibodies can bedetected similarly, e.g., where the purpose is to screen for antibodiesthat body to a BCRP target polypeptide of interest.

The assays are generally carried out under effective conditions. Suchconditions include any environment in which the purpose of the assay canbe accomplished, e.g., to permit specific binding between an antibodyand antigen of interest. These include, suitable antibody and antigenconcentrations; temperature; buffer; the presence of any agent thatfacilitates the interaction, including proteins and other additives toreduce non-specific binding; etc. Specific binding indicates the bindingof a binding partner to a defined amino acid sequence (see above) incontrast to “non-specific binding” to other unintended sequences.

Assays can be carried in liquid or on biological support. For instance,a sample (e.g., blood, stool, urine, cells, stem cells, tissue, cerebralspinal fluid, body fluids, etc.) can be brought in contact with andimmobilized onto a solid phase support or carrier such asnitrocellulose, or other solid support that is capable of immobilizingcells, cell particles or soluble proteins. The support may then bewashed with suitable buffers followed by treatment with a detectablylabeled specific antibody. The solid phase support can then be washedwith a buffer a second time to remove unbound antibody. The amount ofbound label on solid support may then be detected by conventional means.

A “solid phase support or carrier” includes any support capable ofbinding an antigen, antibody, or other specific binding partner.Supports or carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, and magnetite. A support material can have anystructural or physical configuration. Thus, the support configurationmay be spherical, as in a bead, or cylindrical, as in the inside surfaceof a test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads.

One of the many ways in which polypeptide-specific antibody can bedetectably labeled is by linking it to an enzyme and using it in anenzyme immunoassay (EIA). See, e.g., Voller, A., “The Enzyme LinkedImmunosorbent Assay (ELISA),” 1978, Diagnostic Horizons 2, 1-7,Microbiological Associates Quarterly Publication, Walkersville, Md.);Voller, A. et al., 1978, J. Clin. Pathol. 31, 507-520; Butler, J. E.,1981, Meth. Enzymol. 73, 482-523; Maggio, E. (ed.), 1980, EnzymeImmunoassay, CRC Press, Boca Raton, Fla. The enzyme which is bound tothe antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietythat can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes that can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha.-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, .beta.-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods that employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect polypeptides through theuse of a radioimmunoassay (RIA). See, e.g., Weintraub, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March, 1986. The radioactive isotopecan be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wavelength, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Seeabove for others. The antibody can also be detectably labeled usingfluorescence emitting metals such as those in the lanthanide series.These metals can be attached to the antibody using such metal chelatinggroups as diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples of usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

The term “isolated” as used herein indicates that the product is presentin a form which does not naturally occur in nature, e.g., separated,enriched, purified, and otherwise modified. The term “obtainable” asused herein indicates that product can be obtained from the identifiedsource (e.g., monkey), but a product having identical characteristicscan also be produced by other methods, e.g., by recombinant expressionor genetic engineering.

The topic headings set forth above are meant as guidance where certaininformation can be found in the application, but are not intended to bethe only source in the application where information on such topic canbe found.

For other aspects of the polynucleotides, reference is made to standardtextbooks of molecular biology. See, e.g., Hames et al., PolynucleotideHybridization, IL Press, 1985; Davis et al., Basic Methods in MolecularBiology, Elsevir Sciences Publishing, Inc., New York, 1986; Sambrook etal., Molecular Cloning, CSH Press, 1989; Howe, Gene Cloning andManipulation, Cambridge University Press, 1995; Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., 1994-1998.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever. The entiredisclosure of all applications, patents and publications, cited aboveand in the figures are hereby incorporated by reference in theirentirety.

EXAMPLES

Cloning of Rhesus Macaque Breast Cancer Resistance Protein (BCRP)

Full-length sequence was obtained using two strategies. In bothstrategies, the source of total RNA from rhesus pancreas islet cells wasemployed. Total RNA was extracted using standard methods.

1. A fragment of rhesus BCRP was amplified by RT-PCR using a set ofprimers which was known to be able to detect rhesus BCRP (300 bpfragment) (see Sheng Zhou, John D. Shuetz, Kevin D. Bunting, et al. “TheABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cellsand is a molecular determinant of the side-population phenotype. NatureMedicine 2001, vol. 7, 1028, incorporated herein by reference in itsentirety). The amplified fragment was sequenced and used to design GSP 1and GSP2 primers, which were used to perform RACE technology (GSP 1-1:5′-gctgatctccttgaagactgtga-3′ (SEQ ID NO:9): and GSP2-1:5′-atactttgaatcagctggttatc-3′ (SEQ ID NO:10).

SMART RACE cDNA amplification kit (Clontech) was used according to themanufacturer's instructions. In brief, we designed primers GSP1 and GSP2for 5′ RACE and 3′ RACE, respectively, based on our 300 bp cDNA rhesusBCRP product sequences as described above. We synthesized cDNA to theboth ends from this partial sequence using the RACE technology. After aneach round of RACE, an elongated product was submitted for sequencing.Then, a new set of primer was designed each time based on a new sequencefor the further cDNA extension. After 3 rounds of RACE, the full lengthcDNA sequence for rhesus BCRP (SEQ ID NO:1) was determined.

2. The rhesus BCRP cDNA was cloned by RT-PCR using two primers (RhesusBCRP forward: 5′-aaagataaaaactctccagatgtc-3′ (SEQ ID NO:11); and RhesusBCRP reverse: 5′-ttaagaatattttttaagaaataacaat-3′, (SEQ ID NO:12), whichwere designed based on our cDNA sequence generated in (1). The same RNAwas used as described herein.

1. An isolated polypeptide comprising: a) a polypeptide having an aminoacid sequence of SEQ ID NO:2, or a polypeptide fragment thereof, or b) apolypeptide which is encoded by a polynucleotide sequence whichhybridizes to the complement of SEQ ID NO:1 under high stringencyconditions, or a polypeptide fragment thereof, wherein said polypeptideof (a) and (b) is capable of eliciting antibodies which specificallybind to a rhesus monkey BCRP of SEQ ID NO:2.
 2. An isolated polypeptideof claim 1, wherein said polypeptide comprises at least one rhesusmonkey isoform-specific residue selected from the group consisting of:T242, P340, K357, G411, and T615 of SEQ ID NO:2.
 3. An isolatedpolypeptide of claim 1, wherein said polypeptide comprises at least onerhesus monkey-specific residue selected from the group consisting of:M12, T22, R28, V48, G55, P149, V329, D338, I404, G411, N417, F471, T502,I508, V599, and T602 of SEQ IS NO:2.
 4. An isolated polypeptide of claim1, wherein said polypeptide has at least one amino acid substitution atamino acid positions 242, 340, 357, 411, and/or 615 of SEQ ID NO:2,other than the amino acid which occurs in SEQ ID NOS:1 and 5-8.
 5. Anisolated polypeptide of claim 1, wherein said polypeptide has at leastone amino acid substitution at amino acid positions 12, 22, 28, 48, 55,149, 329, 338, 404, 411, 417, 471, 502, 508, 599, and 602 of SEQ IDNO:2, other than the amino acid which occurs in SEQ ID NOS:1 and 5-8. 6.An isolated polypeptide of claim 1, which is obtainable from rhesusmonkey and which comprises T615 of SEQ ID NO:2.
 7. An isolatedpolypeptide of claim 1, which comprises the amino acid sequence setforth in SEQ ID NO:2.
 8. An isolated polypeptide of claim 1, whichconsists of the amino acid sequence set forth in SEQ ID NO:4, or whichconsists essentially of the amino acid sequence set forth in SEQ IDNO:4.
 9. An isolated polypeptide of claim 1, which consists of apolypeptide which is encoded by a polynucleotide sequence whichhybridizes to the complement of SEQ ID NO:3 under high stringencyconditions, or which consists essentially of a polypeptide which isencoded by a polynucleotide sequence which hybridizes to the complementof SEQ ID NO:3 under high stringency conditions.
 10. An isolatedpolypeptide of claim 9, wherein said polypeptide comprises T615 of SEQID NO:2.
 11. A fusion protein which comprises a heterologous peptideamino acid sequence fused to a polypeptide of claim
 10. 12. An isolatedantibody, or fragment thereof, which specifically binds to a polypeptideof claim
 1. 13. An isolated antibody, or fragment thereof, whichspecifically binds to an epitope present in a polypeptide of claim 8,and which is specific for a rhesus monkey BRCP polypeptide.
 14. Anisolated antibody, or fragment thereof, which specifically binds to anepitope present in a polypeptide of claim 9, and which is specific for arhesus monkey BRCP polypeptide.
 15. An isolated antibody, or fragmentthereof, which specifically binds to an epitope present in a polypeptideof claim 9, wherein said epitope comprises T615.
 16. A method ofdetecting a rhesus monkey BCRP polypeptide, comprising contacting asample comprising a rhesus monkey BCRP polypeptide with an antibody ofclaim 12 under conditions effective for said antibody to specificallybind to said polypeptide; and detecting said specific binding of saidantibody to said BCRP polypeptide.
 17. A method of claim 16, whereinsaid cells are stem cells.
 18. A method of claim 16, wherein saiddetecting is accomplished using FACS or an immunoassay.
 19. An isolatedpolynucleotide coding for a polypeptide of claim
 1. 20. An isolatedpolynucleotide comprising a) a polynucleotide comprising the nucleotidesequence set forth in SEQ ID NO:1; b) a polynucleotide comprising thenucleotide sequence set forth in SEQ ID NO:3; c) a polynucleotidecomprising a nucleotide sequence coding for the amino acid sequence setforth in SEQ ID NO:2; d) a polynucleotide sequence comprising anucleotide sequence which hybridizes to the complement of SEQ ID NO:1under high stringency conditions and which codes for a polypeptidehaving an ATPase and drug transport activity.